The application of high-throughput screening (HTS) technologies for the discovery and development of new therapeutic drugs is now well established within the pharmaceutical industry. In the HTS process, drug candidates are screened for possible effects in biological systems and for the specificity of selected lead compounds towards particular targets. Primary screening has been addressed by the development of HTS assay processes and assay miniaturisation utilising the microtitier well plate format with 384, 864, 1536 or greater miniaturised wells and are capable of allowing throughput levels of over 100,000 tests/day in primary screening. Lead compounds identified during the primary screening process are then required to undergo further refined screening and testing in a variety of assays in order to investigate the biological compatibility of the compound. Such assays may include receptor binding and enzyme activity assays, in addition to bioavailability, metabolism and toxicology. Secondary screening of lead compounds can identify potentially undesirable side effects and/or secondary therapeutic activities not identified in the primary screening process and these assays are carried out predominantly using cultured cell lines. In comparison with assays used in the primary screening process, secondary screening assays have a much higher level of complexity and more stringent requirements, both in the mechanics of the assay and in the information generated.
The detection of in vitro binding events, such as receptor binding assays, enzyme assays and immunoassays using scintillation proximity assays (SPA) is now an established technology and is used in HTS applications (Cook, N. D., Drug Discovery Today, Vol 1 (7), (1996), 287–294). SPA utilises scintillant-containing microspheres to which ligands (eg. antibodies, binding proteins, etc) have been attached. When a radioisotopically labelled molecule is brought into close proximity to the scintillant in the microsphere, energy transfer from the radioisotopic decay takes place, resulting in the emission of light. Any radioisotope remaining free in solution, will dissipate its energy into the aqueous medium and will remain undetected. SPA has also been applied to the study of cellular biochemical processes in situ, using cultured living cells. European Patent No. 650396 discloses a method and an apparatus for studying a cellular process, for example, a microwell plate. Each well of the microwell plate includes a scintillant layer in the base, which is further treated to facilitate the attachment and/or growth of cells. In an alternative format, the device may be a single well or tube which is composed of a non-scintillant containing material, into which is placed a circular, scintillant-containing plastic disc. The method for studying a cellular process includes culturing cells adhering to the scintillant layer, in the presence of a fluid medium, introducing into the fluid medium a reagent labelled with a radioisotope emitting electrons, such that a portion of the labelled reagent becomes associated with or released from the cells adhering to the layer. Scintillation events caused by the proximity of the radiolabelled reagent to the scintillant containing base are detected so as to study the cellular process.
PCT Application No. WO97/40189 relates to a method for quantifying the amount of target nucleic acid such as mRNA in morphologically intact cells, the method comprising the steps of culturing not less than two physically distinct samples of cells on at least one substrate, contacting the cells with a fixative and exposing the fixed cells to a labelled nucleic acid probe to hybridise with the target nucleic acid sequence. This invention is concerned therefore with measurements of hybridisation of nucleic acid probes to fixed cells following processing and washing. The method of the invention does not describe and is not compatible with measurement of dynamic processes in living cells.
PCT Application No. WO 96/19739 describes a solid support for use in radioligand binding assays, the support comprising a plurality of interconnected elements arranged to provide interstitial spaces in which a liquid can flow. The support comprises plastic beads which, in a preferred form may contain a scintillant, and which are fused together to form a solid support for in vitro binding assays. While the solid support may also be used for cell growth, it is stated that the support should preferably not contain a fluorophore.
The use in cell culture of CYTODEX™ Microcarrier support particles (Amersham Pharmacia Biotech) has improved the yields of anchorage dependant cells by increasing the surface area for growth. Properties of these microcarriers include optimised size and density for maximum cell growth, a biologically inert matrix that provides a strong but non-rigid substrate for stirred cultures and transparency for easy microscopic examination of attached cells. Microcarriers can be used in either suspension cultures or monolayer cultures to increase the surface area of the culture vessels and perfusion chambers. The increased surface area allows enables the production of increased densities of cells, viruses and cell products.